One example of illumination used in detection systems is fluorescence, and an example of the use of fluorescence detection is in nucleic acid testing (NAT). This is a core element in molecular diagnostics for detecting genetic predispositions for diseases, for determining RNA expression levels or identification of pathogens, like bacteria and viruses that cause infections.
The detection of fluorescence can be used both for a qualitative determination of the presence of a particular target DNA sample, and for a quantitative determination of the amount of DNA present in a sample. This invention relates to the apparatus used to detect the fluorescence, and the method of use.
In a typical molecular diagnostic experiment, a bio-sample is screened for detection of certain biological components (the “target”), such as genes or proteins. This is done by detecting the occurrence of selective bindings (known as hybridization) of the target to a capture probe, which is attached to a solid surface. The hybridization step is typically followed by a washing step, where all unbounded target molecules are flushed away, and finally a detection step is carried out.
The detection is based on fluorescent detection of fluorescent labels attached to the target molecules. The fluorescent detection needs to be very sensitive, and surface specific so as to minimize the biological background. Ideally, the fluorescent detection needs to be capable of single fluorescent label detection, while the process is kept time effective.
In the near future, the detection needs to be performed outside the hospitals or laboratories set up for diagnostics. This requires that the devices are capable of sensitive measurement in relatively short time.
One of the limitations of fluorescence detection in biosensors is this biological background signal. In a typical experiment, fluorescence of molecules bound to a surface needs to be detected. However, the bio-components in the vicinity of the surface can generate a large background signal. Hence relatively long measurement times are needed to compensate with the background. A standard method to mitigate this problem is to use confocal filtering, so that the excitation from a small volume is imaged onto the detector used.
However, a problem with the use of a confocal optical system is again the length of the detection process, as the spot focus needs to be scanned across the area of interest of the sample in order to ensure reliability and sensitivity.
One proposed solution to this problem is to implement line scanning fluorescence detection. A focused line is scanned across the sample, so that the detection time can be reduced. However, the confocal filtering is not optimal in the direction along the axis of the line of excitation. This has the direct result that the detected surface fluorescence is affected by a relatively large background signal and the sensitivity is reduced.